【作者】 李军果；

【导师】 李力；

【作者基本信息】 第三军医大学 ， 妇产科学， 2008， 博士

【摘要】 背景与目的抑癌基因INK4a在多种肿瘤中阴性表达,但在几乎100%宫颈癌中过表达,究其发生原因,目前尚无定论。人乳头瘤病毒（human papilloma virus, HPV）感染是宫颈癌发生的必要条件,其中50%以上是HPV16型。HPV的E6和E7基因整合到宫颈上皮细胞并持续表达是引发宫颈癌的主要原因,同时也是维持宫颈癌恶性表型的必要条件。宫颈癌INK4a过表达是否与HPV感染有关,目前尚无统一认识。本研究拟通过RNA干扰技术,以抑制HPV16E7表达,进而检测对SiHa和CaSki细胞INK4a基因表达的影响;同时,比较不同HPV感染状态的宫颈癌细胞CaSki（HPV16感染）,HeLa（HPV18感染）,C-33A（无HPV感染）中INK4a表达水平,综合分析宫颈癌INK4a过表达与HPV感染的相关关系,初步阐明INK4a基因在宫颈癌中异常表达的可能机制,为宫颈癌的预防和早期诊断提供理论和实验依据。方法1.应用免疫组织化学技术检测126例HPV感染及未感染宫颈癌（cervical cancer,CC）、宫颈上皮内瘤变（cervical intraepithelial neoplasia,CIN）及慢性宫颈炎中INK4a表达规律及其与HPV感染之间的相互关系2.利用RNA干扰技术,针对HPV16E7的siRNA干涉SiHa和CaSki细胞,使HPV16E7 mRNA沉默3.应用RT-PCR技术,检测HPV16E7 siRNA干扰前后SiHa和CaSki细胞HPV16E7及INK4a mRNA4.应用Western blot技术,检测HPV16E7 siRNA干扰前后SiHa和CaSki细胞HPV16E7及INK4a蛋白表达5.应用流式细胞仪,检测HPV16E7 siRNA干扰前后SiHa和CaSki细胞周期分布情况6.联合应用RT-PCR和Western blot技术,比较不同HPV感染状态的宫颈癌细胞CaSki（HPV16感染）,HeLa（HPV18感染）,C-33A（无HPV感染）中INK4a表达水平结果1. 126例宫颈病变组织中INK4a表达规律及其与HPV感染之间的关系如下:（1） 126例宫颈病变组织中,CC,CIN,慢性宫颈炎中p16^INK4a阳性表达率分别为100.00（50/50）、71.93（41/57）、42.11（8/19）,三者间差异有显著性意义（p<0.05）。（2） 89例hr-HPV（+）宫颈病变组织中,CC,CIN,慢性宫颈炎中p16^INK4a阳性表达率分别为100.00%（42/42）、76.32%（29/38）、44.44%（4/9）,三者间差异有显著性意义（p<0.05）。（3） 37例hr-HPV（-）宫颈病变组织中,CC,CIN,慢性宫颈炎中p16^INK4a阳性表达率分别为100.00%（8/8）、63.16%（12/19）、40.00%（4/10）,三者间差异有显著性意义（p<0.05）。（4） 57例CIN中,CIN1,CIN2,CIN3 p16^INK4a阳性表达率分别为36.84%（7/19）、81.82%（18/22）、100.00%（16/16）,且三者间差异有显著性意义（p<0.05）。（5） 38例hr-HPV（+）CIN中,CIN1,CIN2,CIN3 p16^INK4a阳性表达率分别为37.50%（3/8）、73.33%（11/15）、100.00%（15/15）,CIN3与CIN2和CIN1之间差异均有显著性意义（p<0.05）,但CIN2和CIN1之间差异无显著性意义（p>0.05）。（6） 107例CC和CIN组织中p16^INK4a表达和hr-HPV DNA检测同时阳性71例,占66.36%（71/107）;p16^INK4a阳性表达而hr-HPV（-）21例,占19.63%（21/107）;p16^INK4a阴性表达而hr-HPV（+）8例,占7.57%（8/107）;p16^INK4a表达或hr-HPV DNA检测单一阳性加上共阳性占93.46%（100/107）。Pearson相关分析CC和CIN中p16^INK4a表达与宫颈hr-HPV感染相互间无相关性（p>0.05）。（7） 47例CC和16例CIN3中p16^INK4a全部阳性表达,无论HPV感染状态如何;38例hr-HPV（+）CIN组织中p16^INK4a表达29例,占76.32%（29/38）;19例hr-HPV（-）CIN组织中p16^INK4a表达12例,占63.16%（12/19）,两者间差异没有显著性意义（p>0.05）;15例hr-HPV（+）CIN2组织中p16^INK4a表达11例,占73.33%（11/15）;7例hr-HPV（-）CIN2组织中p16^INK4a表达7例,占100.00%（7/7）,两者间差异没有显著性意义（p>0.05）;8例hr-HPV（+）CIN1组织中p16^INK4a表达3例,占37.50%（3/8）;11例hr-HPV（-）CIN1组织中p16^INK4a表达4例,占36.36%（4/11）;两者间差异没有显著性意义（p>0.05）;9例hr-HPV（+）慢性宫颈炎中p16^INK4a表达4例,占44.44%（4/9）;10例hr-HPV（-）慢性宫颈炎中p16^INK4a表达4例,占40.00%（4/10）;两者间差异没有显著性意义（p>0.05）。比较分析发现,p16^INK4a蛋白在hr-HPV（+）和hr-HPV（-）宫颈病变组织中阳性表达率差异无显著性意义（p>0.05）。2. siHPV16E7 001,siHPV16E7 002,siHPV16E7 003干扰CaSki细胞后,HPV16E7 mRNA的含量下降,其中siHPV16E7 001干扰后,HPV16E7 mRNA的含量下降最明显,约80%;不同浓度siHPV16E7 001干扰CaSki细胞后,60nM组干扰效率最强,与未干扰组比较差异有显著性意义（p<0.01）,且细胞毒性轻微。3.和SiHa-Nontransfection组（0.6033±0.0186）及SiHa-siNControl组（0.5500±0.0173）相比,在SiHa-siHPV16E7组（0.3067±0.0120）中,RT-PCR方法证明其HPV16E7 mRNA表达显著降低（p<0.01）,Western blot方法亦证明SiHa-siHPV16E7组的HPV16E7蛋白表达量显著低于SiHa-Nontransfection组和SiHa-siNControl组（p<0.01）。提示通过RNA干扰方法能有效地抑制SiHa细胞的HPV16E7表达。4.和CaSki-Nontransfection组（1.1733±0.0667）及CaSki-siNControl组（1.3933±0.1667）相比,在CaSki-siHPV16E7组（0.4033±0.0328）中,RT-PCR方法证明其HPV16E7 mRNA表达显著降低（p<0.01）,Western blot方法亦证明CaSki-siHPV16E7组的HPV16E7蛋白表达量显著低于CaSki-Nontransfection组和CaSki-siNControl组（p<0.01）。提示通过RNA干扰方法能有效地抑制CaSki细胞的HPV16E7表达。5.和SiHa-Nontransfection组（1.0033±0.0233）及SiHa-siNControl组（0.9267±0.0371）相比,在SiHa-siHPV16E7组（0.5833±0.0133）中,RT-PCR方法证明其INK4a mRNA表达显著降低（p<0.01）,Western blot方法亦证明SiHa-siHPV16E7组的INK4a蛋白表达量显著低于SiHa-Nontransfection组和SiHa-siNControl组（p<0.01）。提示通过siHPV16E7干扰能有效地抑制SiHa细胞的INK4a表达。6.和CaSki-Nontransfection组（0.7833±0.0088）及CaSki-siNControl组（0.8533±0.0353）相比,在CaSki-siHPV16E7组（0.4767±0.0145）中,RT-PCR方法证明其INK4a mRNA表达显著降低（p<0.01）,Western blot方法亦证明CaSki-siHPV16E7组的INK4a蛋白表达量显著低于CaSki-Nontransfection组和CaSki-siNControl组（p<0.01）。提示通过siHPV16E7干扰能有效地抑制CaSki细胞的INK4a表达。7.细胞周期分析结果表明:HPV16E7干扰SiHa细胞24h,48h,72h后,G0-G1期细胞百分数较SiHa-Nontransfection组依次增加了1.1%,0.7%,31.7%,进入S期的细胞百分数较SiHa-Nontransfection组依次减少了0.2%,2.5%,6.4%;HPV16E7干扰CaSki细胞24h,48h,72h后,G0-G1期细胞百分数较CaSki-Nontransfection组依次增加了8.7%,12.7%,15.6%,进入S期的细胞百分数较CaSki-Nontransfection组减少了1.3%,4.3%,14.9%。提示通过siHPV16E7干扰能将更多的细胞阻滞在G0-G1期。8. RT-PCR检测不同HPV感染状态的宫颈癌细胞CaSki（0.3967±0.0491） ,HeLa（0.5567±0.1037）,C-33A（0.5633±0.0722）中INK4a mRNA表达无明显差异（p>0.05）;Western blot方法亦证明CaSki（0.4152±0.0255） , HeLa（0.5094±0.0361） , C-33A （0.5895±0.0409）中INK4a蛋白表达无显著性差异（p>0.05）。提示不同HPV感染状态的宫颈癌细胞中INK4a表达规律趋于一致。结论1.无论是否合并HPV感染,宫颈癌及CIN3组织中INK4a基因都过表达。2. siHPV16E7可序列特异性下调宫颈癌SiHa,CaSki细胞HPV16E7基因水平,显著降低HPV16E7蛋白表达。3.通过RNA干扰抑制HPV16E7表达后,能显著抑制INK4a基因表达,同时将更多的细胞阻滞在G0-G1期。4.不同HPV感染状态的宫颈癌细胞中INK4a表达规律趋于一致。综上提示,在合并HPV感染的宫颈癌中INK4a基因过表达与HPV感染有关;未合并HPV感染的宫颈癌中INK4a基因同样过表达,应另有其他机制存在。检测INK4a基因表达,对于宫颈癌的早期诊断和筛查具有非常有价值的临床意义。更多还原

【Abstract】 Background and Objective Tumor suppressor gene INK4a encodes p16^INK4a protein and aberrant expression of p16^INK4a protein has been reported in various malignancies, whereas p16^INK4a overexpression has been detected in almost all precancerous and cancerous lesions of the cervix and it’s reason has been indefinite. Human papilloma virus（HPV） infection was essential for genesis of cervical carcinomas. About 50% of cervical cancers was related with HPV type 16（HPV16）.After infection, E6 and E7 gene of HPV were integrated to genome of cervical epithelium. Continued expression of transforming oncoprotein E6 and E7 not only drives the neoplastic progression in cervical epithelium but also plays important role in maintaining malignant phenotype of cervical cancer cells. Correlations between p16^INK4a overexpression and HPV infection has no accepted argument. This study was carried out to assess the correlations between p16^INK4a expression and the HPV infection by HPV16E7 gene silencing using small interfering RNA（siRNA） in human cervical carcinomas cell lines SiHa and CaSki and by comparing p16^INK4a expression in CaSki（HPV16 infection）, HeLa（HPV18 infection）, C-33A（no HPV infection）; to ascertain the role of p16^INK4a immunostaining as an early biomarker of uterine cervix carcinomas.Methods1. The expression of p16^INK4a protein was detected in 126 cervical lesions samples by immunohistochemical staining, including cervical cancer（CC）,cervical intraepithelial neoplasia（CIN） and cervicitis with high-risk Human papillomavirus（hr-HPV） infected or no, and to investigate the relationships of p16^INK4a protein expression with hr-HPV state.2. RNA interference（RNAi） originated by chemically synthetic small interference RNA （siRNA） could induce HPV16E7 gene silencing in cervical carcinomas cell lines SiHa and CaSki.3. Expressions of HPV16E7 and INK4a mRNA were examined by semiquantitative reverse transcription polymerase chain（RT-PCR） in SiHa-Nontransfection, SiHa -siHPV16E7, SiHa-siNControl and CaSki-Nontransfection, CaSki-siHPV16E7,CaSki-siNControl cells respectively.4. Expressions of HPV16E7 and INK4a protein were examined by Western blot in SiHa-Nontransfection, SiHa-siHPV16E7, SiHa-siNControl and CaSki-Nontransfection, CaSki-siHPV16E7, CaSki-siNControl cells respectively.5. Cell cycle analysis were assessed by flowcytometry（FCM）in SiHa-Nontransfection, SiHa-siHPV16E7 and CaSki-Nontransfection, CaSki-siHPV16E7, cells respectively.6. Expressions of INK4a mRNA and protein were examined by RT-PCR and Western blot in CaSki, HeLa and C-33A cells with or without HPV16E7 infection,respectively.Results1. Immunostaining for p16^INK4a and relationship between p16^INK4a immunoreactivity and HPV in 126 cervical lesions samples: （1） The positive expression rates of p16^INK4a in CC,CIN and cervicitis samples were 100.00（50/50）, 71.93%（41/57） and 42.11%（8/19） respectively, and there was obvious difference between the three groups（p<0.05）. （2）The positive expression rates of p16^INK4a in CC,CIN and cervicitis samples（n=89）with HPV infection were 100.00%（42/42）, 76.32%（29/38） and 44.44%（4/9） respectively, and there was obvious difference between the three groups（p<0.05）. （3） The positive expression rates of p16^INK4a in CC,CIN and cervicitis samples（n=37） without HPV infection were 100.00% （8/8）, 63.16%（12/19） and 40.00%（4/10） respectively, and there was obvious difference between the three groups（p<0.05）. （4） The positive expression rates of p16^INK4a in CIN1,CIN2 and CIN3 samples（n=57） were 36.84%（7/19）, 81.82%（18/22） and 100.00%（16/16）, respectively, and there was obvious difference between the three groups（p<0.05）. （5） The positive expression rates of p16^INK4a in CIN1,CIN2 and CIN3 samples（n=57） with HPV infection were 37.50%（3/8）, 73.33%（11/15） and 100.00%（15/15） respectively, and there was obvious difference between CIN3 and CIN2 or CIN1, but there was no obvious difference between CIN2 and CIN1（p>0.05）. （6） In 107 CC and CIN, 71（66.36%） cases showed simultaneous positive expression of p16^INK4a proteins and hr-HPV DNA, 21（19.63%） cases showed positive expression of p16^INK4a and negative expression of hr-HPV DNA, 8（7.57%） cases showed negative expression of p16^INK4a and positive expression of hr-HPV DNA, the positive rates of p16^INK4a and/or hr-HPV DNA expressions was 93.46%, whereas there was no significant relationship between p16^INK4a expression and hr-HPV DNA infection（p>0.05）. （7） p16^INK4a protein was positive in all of CC（n=47） and CIN3（n=16） cases with or without HPV infection. The positive expression rates of p16^INK4a in hr-HPV（+） and hr-HPV（-） CIN samples were 76.32%（29/38） and 63.16%（12/19） respectively, and there was no obvious difference between the two groups（p<0.05）, as well as in hr-HPV（+） and hr-HPV（-） cervicitis tissues, the positive rates of p16^INK4a expression were 44.44%（4/9） and 40.00%（4/10） respectively. This expression increases from cervicitis, CIN to CC, and the significant difference was observed between the groups of CC, CIN and cervicitis with hr-HPV infected or no（p<0.05）.2. When CaSki cells were interfered by siHPV16E7 001,siHPV16E7 002 and siHPV16E7 003, the levels of mRNA encoding HPV16E7 in cells was reduced, especially siHPV16E7 001 can make HPV16E7 mRNA reduce by 80%. Moreover, the most suitable multiplicity of infection of siHPV16E7 001 for CaSki cells was 60nM.3. Compared with SiHa-Nontransfection（0.6033±0.0186） and SiHa-siNControl （0.5500±0.0173）, HPV16E7 mRNA expression（0.3067±0.0120） surveyed by RT-PCR decreased markedly in SiHa-siHPV16E7 group（p<0.01）, and similar alterations were found in HPV16E7 protein expression among these three groups. The above mentioned results suggested that HPV16E7 expression in SiHa cells could be inhibited efficiently by RNAi.4. Compared with CaSki-Nontransfection（1.1733±0.0667） and CaSki-siNControl （1.3933±0.1667）, HPV16E7 mRNA expression（0.4033±0.0328） surveyed by RT-PCR decreased markedly in CaSki-siHPV16E7 group（p<0.01）, and similar alterations were found in HPV16E7 protein expression among these three groups. The above mentioned results suggested that HPV16E7 expression in CaSki cells could be inhibited efficiently by RNAi.5. Compared with SiHa-Nontransfection（1.0033±0.0233） and SiHa-siNControl （0.9267±0.0371）, INK4a mRNA expression（0.5833±0.0133） surveyed by RT-PCR decreased markedly in SiHa-siHPV16E7 group（p<0.01）, and similar alterations were found in INK4a protein expression among these three groups. The above mentioned results suggested that INK4a expression in SiHa cells could be inhibited efficiently by siHPV16E7 interference.6. Compared with CaSki-Nontransfection（0.7833±0.0088） and CaSki-siNControl （0.8533±0.0353）, INK4a mRNA expression（0.4767±0.0145） surveyed by RT-PCR decreased markedly in CaSki-siHPV16E7 group（p<0.01）, and similar alterations were found in INK4a protein expression among these three groups. The above mentioned results suggested that INK4a expression in CaSki cells could be inhibited efficiently by siHPV16E7 interference.7. Cell cycle analysis showed that siHPV16E7 induced accumulation of SiHa and CaSki cells in G0-G1 phase by 31.7% and 15.6% with a decrease in the percentage of cells in S-phase by 6.4% and 14.9% relative to control. These sequence specific siRNAs showed a blockbuster effect in arrestment of the cell cycle.8. Compared with CaSki（0.3967±0.0491） and HeLa（0.5567±0.1037）, INK4a mRNA expression（0.5633±0.0722） surveyed by RT-PCR had no obvious difference in C-33A cells （p>0.05）, and similar alterations were found in INK4a protein expression among these three groups. The above mentioned results suggested that INK4a expression was at equal pace in CC cells with or without HPV infection.Conclusion1. Overexpression of INK4A gene in precancerous and cancerous lesions of the cervix with or without HPV infection.2. These sequence specific siRNAs showed a blockbuster effect in downregulation of HPV16E7 gene expression in CC cells SiHa and CaSki.3. INK4a expression in SiHa and CaSki cells could be inhibited efficiently by siHPV16E7 interference,moreover, these sequence specific siRNAs showed a blockbuster effect in arrestment of the cell cycle.4. INK4a expression was with one accord in CC cells with or without HPV infection. Therefore/ In a word, the above mentioned conclusion suggested that INK4a expression was at equal pace in CC cells with or without HPV infection. Overexpression of p16 INK4a protein is associated with HPV in cervical cell carcinoma and dysplasia with HPV infection,whereas there are else mechanisms on overexpression of p16 INK4a in CC and dysplasia without HPV infection. Overexpression of INK4a gene help to define the screening and early diagnosis of cervical cancer cases.更多还原